Apoptosis plays a central role in the development and homeostasis of all multi-cellular organisms. Abnormal inhibition of apoptosis is a hallmark of cancer and autoimmune diseases, whereas excessive activation of cell death is implicated in neuro-degenerative disorders such as Alzheimer's disease. Pro-apoptotic chemotherapeutic drugs provide a recent approach to overcoming the clinical problem of drug resistance. See, e.g., Makin, et al., Cell Tissue Res. 301:143-52 (2000).
The mechanism of apoptosis is conserved across species and executed with a cascade of sequential activation of proteases called caspases. Once activated, these caspases are responsible for proteolytic cleavage of a broad spectrum of cellular targets that ultimately lead to cell death. IAPs (inhibitor-of-apoptosis proteins) regulate apoptosis by inhibiting caspases; and a protein called Smac (“Smac” stands for second mitochondria-derived activator of caspases, and is a mitochondrial protein) binds to and inhibits IAPs, and thereby promotes caspase activation.
Defective apoptosis regulation can confer resistance to many current treatment protocols, leading to tumor growth. This may occur as a result of overexpression of IAPs, which inhibit the caspases that would otherwise initiate apoptosis. Alternatively, deregulation can occur as a result of underproduction of the Smac peptides that act to inhibit IAP activity. Deficiency of Smac can thus allow IAP to prevent apoptosis from occurring when it should, and a Smac mimetic like the present compounds can replace the activity of Smac and thus promote desired apoptosis.
Debatin, et al., WO 03/086470, describes Smac-peptides as therapeutic agents useful against cancer and autoimmune diseases; they are reported to act by sensitizing the cells toward TRAIL-induced or anticancer drug-induced apoptosis. (TRAIL stands for TNFα related apoptosis-inducing ligand). See also Li, et al., Science 305:1471-1474 (2004). Debatin provides in vivo evidence that Smac induces the eradication of certain tumors such as glioblastoma tumor models in animals when administered in combination with TRAIL. According to Debatin, aggressive cancer phenotypes, which result from deregulation of signaling pathways, commonly fail to undergo apoptosis when they otherwise would, allowing rapid and abnormal tissue growth. Bockbrader, et al., disclose efficacy of Smac mimic compounds on breast cancer cell lines when used in conjunction with TRAIL or etoposide, or when used in cells that express TRAIL at relatively high levels. Oncogene 24:7381-7388 (2005).
Similarly, according to Debatin, defects in apoptosis regulation play a key role in the pathogenesis of autoimmune disorders, including lupus erythematodes disseminatus and rheumatoid arthritis. Accordingly, compounds that mimic the activity of Smac can treat some of the effects of such conditions.
The protein Smac has been shown to inhibit a wide variety of IAPs, and is believed to be a key regulator of apoptosis in mammals. See Du, et al., Cell 102:33-43 (2000); Verhagen et al., Cell 102:43-53 (2000); and Vucic et al., Biochem. J. 385(1):11-20 (2005). N-terminal Smac-derived peptides and mimetics have been shown to similarly inhibit IAPs, and promote caspase-8 activation. See Petersen, et al., Cancer Cell 12:445-456 (2007); Varfolomeev, et al., Cell 131:669-681 (2007); and Vince, et al., Cell 131:682-693 (2007).
These studies further showed that TNFα, a death receptor ligand, is the major actor in cancer cell apoptosis induced by Smac mimetics. IAPs are components of TNFR (tumor necrosis factor receptor), so IAP inhibitors can divert TNFR signaling from an NF-κB-mediated pro-inflammatory signal, to an anti-inflammatory apoptotic signal. Acute reduction in these IAPs activates the NF-κB pathways and causes autocrine TNFα production, which in turn leads to TNFR occupancy, caspase-8 activation, and cell death.
Eukaryotic DNA is packaged into chromatin which appears as a series of “beads on a string,” with the beads being the individual nucleosomes. Kornberg & Lorch, Cell 98:285-294 (1999). Each nucleosome consists of eight core histone proteins (two each of H3, H4, H2A and H2B), which are wrapped by 147 base pairs of DNA in a left-handed superhelix, forming the intact nucleosome. Luger, et al., Nature 389:251-260 (1997).
Several factors, including DNA methylation, histone modifications, and small nuclear RNAs, have been implicated in the regulation of transcription from chromatin, and are referred to as “epigenetic regulations.” Epigenetic mechanisms are essential for development, cell differentiation, and protection against viral genomes, and seem to be critical for the integration of endogenous and environmental signals during the life of a cell or an organism. By analogy, deregulation of epigenetic mechanisms has been associated with a variety of human diseases, most notably cancer.
Different types of epigenetic modifications are closely linked and often act in self-reinforcing manner in the regulation of different cellular processes. DNA methylation and histone acetylation are major epigenetic modifications that are dynamically linked in the epigenetic control of gene expression and their deregulation plays an important role in tumorigenesis. See Feinberg, et al., Nat. Rev. Genet. 7:21-33 (2006); Jones & Baylin, Nat. Rev. Genet. 3:415-428 (2002). Recent studies suggested that an intimate communication and mutual dependence exists between histone acetylation and DNA methylation in the process of gene silencing. Communication between histone acetylation and cytosine methylation may proceed in both directions. In one scenario, DNA methylation may be the primary mark for gene silencing that triggers events leading to non-permissive chromatin state. In another scenario, the loss of histone acetylation may serve as the initial event of gene silencing, which is followed by DNA methylase targeting and induction of local DNA hypermethylation. See Vaissiere, et al., Mut. Res. 659:40-48 (2008).